Sh nc

To stably knock down FOXC1, HOTTIP, or Sp1 expression, we purchased lentiviral transduction particles expressing FOXC1 shRNA (shFOXC1), Sp1 shRNA (shSp1), or control shRNA (shNC) from Sigma‐Aldrich (St Louis, MO, USA). For HOTTIP shRNA (shHOTTIP), we designed four shRNA sequences shHOTTIP #1: 5′‐AAA AGC ATC TCA AAT TAA GCT TTG CCT CGA GGC AAA GCT TAA TTT GAG ATG C‐3′; shHOTTIP #2: 5′‐AAA AGG GAC TGA ATT CTT CGA GAT TTC TCG AGA AAT CTC AAG AAT TCA GTC CC‐3′; shHOTTIP #3: 5′‐AAA AGG TGC ACC TTA TTG ATC AAA TCT CGA GAT TTG AAT AAG GTG CAC C‐3′; shHOTTIP #4: 5′‐AAA AGC AGC CAA CAA ACT GAC TTG CCT CGA GGC AAG TCA GTT TGT TGG CTG C‐3′), cloned them into OIP5 shRNA lentiviral plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and generated lentiviral particles. Preliminary experiments in 143B and SW1353 cells showed that shHOTTIP #1 and #2 presented the most robust knockdown effect and thus were used for this study. Puromycin (2.5 mg·mL⁻¹; Sigma‐Aldrich) was applied for 7 days to select for stable cells.
To transiently target the expressions of EZH2, or LSD1, two distinct siRNAs for each specific target gene were purchased from Dharmacon (Lafayette, CO, USA) and transfected into 143B or SW1353 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). siRNA targeting no known human or mouse genes (si‐NC) was used in parallel.

A total of 24 female 4-week BALB/c nude mice purchased from Beijing Institute of Pharmacology and Toxicology, Chinese Academy of Medical Sciences were selected in this study, and individually housed in specific pathogen-free animal laboratory at 22–25°C, humidity of 60% – 65%, and a 12-h light/12-h dark cycle with free access to food and water. After one week of adaptive feeding, mice were subjected to subsequent experiments.
Lentivirus harboring short hairpin RNA (shRNA) against lncRNA PVT1 (sh-PVT1), sh-p53 or sh-NC (Sigma-Aldrich) was titrated to 10⁹ TU/mL. U373 cells were seeded in a 6-well plate at 2 × 10⁵ cells/well and cultured for 24 h. U373 cells were infected with lentivirus for 72 h and cultured at least 14 days in medium containing 4 μg/mL puromycin. Cells resistant to puromycin were cultured for 9 days in medium containing 2 μg/mL puromycin and further cultured in puromycin-free medium to collect U373 cells with stable knockdown of lncRNA PVT1 or p53.
Nude mice were injected with 2 × 10⁶ U373 cells that had been transfected with plasmids of shRNA-p53, shRNA-PVT1, or both at the armpit of upper limb. The diameter of tumor was measured twice every 8 days. Thirty two days later, mice were euthanized, after which tumor volume and weight were assessed.

MALAT1 and coding sequences of LIN28A and Nox4 were cloned into the pcDNA3.1 vector (V79020, ThermoFisher, Waltham, MA, USA) for overexpression of MALAT1 (oe-MALAT1), LIN28A (oe-LIN28A) and Nox4 (oe-Nox4). siRNA (10 nM) was selected for transient transfection thanks to its easy generation and introduction into cells with high efficiency. Specially, si-RNAs against MALAT1 (si-MALAT1), LIN28A (si-LIN28A) and Nox4 (si-Nox4) and scrambled negative control siRNAs (si-NC) were purchased from RiboBio (Guangzhou, China). HK-2 cells were transfected with vector, oe-MALAT1, si-MALAT1, oe-LIN28A, si-LIN28A, oe-Nox4, si-Nox4, si-LIN28A+vector, si-LIN28A+oe-Nox4, si-MALAT1+vector, si-MALAT1+oe-LIN28A or si-NC using the Lipo3000 reagent (L3000015, ThermoFisher) following the manual. Cells were harvested at 48 hours after transfection for subsequent assays. In some assays, cells were harvested and treated with NG or HG. For in vivo knockdown of MALAT1, viral vector-based shRNA with high transduction efficiency and long-term effect was used. Specially, the shRNA against MALAT1 (sh-MALAT1, Sigma) and scrambled shRNA (sh-NC, Sigma) were inserted into the pLKO.1 lentiviral vector (10878, Addgene, Watertown, MA, USA), sh-MALAT1 and sh-NC lentiviral particles were packaged in HEK-293T cells.

Plasmids including pCMV-Flag-His-puro-MELK (MELK), pCMV-Flag-His-puro-FOXM1 (FOXM1) and the empty vector pCMV-Flag-His-puro (Vector) were purchased from Transheep (Shanghai, China). For MELK overexpression, cells were transfected with 2 μg plasmids (MELK or Vector) for 48 h by using the Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Inc.) following the manufacturer's manual (23 (link)). Cells were then selected in the presence of 1.5 μg/mL puromycin (Sigma-Aldrich). After selection for 3 weeks, the stable colonies were picked up and expanded. For ectopic expression of FOXM1, the MELK-silenced KYSE30 and EC9706 cells were transfected with 2 μg plasmids (FOXM1 or Vector) for 48 h by using the Lipofectamine 2000 reagent. The cells were then exposed to puromycin (1.5 μg/mL) for 3 weeks.
Specific shRNAs targeting MELK (shMELK#1 and shMELK#2) or FOXM1 (shFOXM1#1 and shFOXM1#2) and a scramble shRNA (shNC) were obtained from Sigma-Aldrich. The indicated sequences were described in Table S1. Lentiviruses production and subsequently tranfection were performed as our previously described (23 (link)). The cells were then cultured with puromycin for 3 weeks to establish stable cells.

Three designs of shRNAs targeting ENaCα and the scrambled non-coding shRNAs (shNC) were purchased from (Sigma-Aldrich) (Table 1). To package the shRNAs into the lentivirus, envelope vector pMD2.G (Addgene, 12259), packaging vector psPAX2 (Addgene, 12260) and shRNAs together with lipofectamine 2000 (ThermoFisher Scientific) were transfected into 293FT cells. Packaged lentivirus was harvested 72 h after transfection and next transfected into ISK or HBE cells for 24 h in the presence of polybrene (6 μg/ml). The cells were then cultured in the presence of puromycin (1 μg/ml) for 14 days to achieve stable knockdown.